Optical scanning microscopes, frequently also referred to as "laser scan microscopes", have been known for a long time. For example, the "Journal of Microscopy", Vol. 138, Pt 1, (Apr. 1985on pages 29-34, describes an optical scanning microscope of confocal construction which effects the scanning of the object by means of a moving table. The optical system of this microscope is stationary and is designed to focus the smallest possible light spot which can be produced with limited diffraction in the object plane. A condenser, in the form of a second objective, serves to collect the light which passes through the object. A photomultiplier is positioned behind said objective (after corresponding deflection of the beam), and the signals of the photomultiplier are used to create the object image on a monitor.
A scanning optical microscope of a somewhat different construction is shown in the Carl Zeiss publication W41-910e, "Laser Scan Microscope: An Optical Scanning Microscope", printing annotation IX/84. In this scanning microscope, the laser beam, which is used for the scanning, is itself moved over the stationary object. The construction of this scanning optical microscope is shown in FIG. 1 and will be described further below with reference to said figure.
In conventional microscopes, "Zernicke phase contrast" is a contrast method which has been used for a long time. The theory and practice of this method are, for example, described in great detail in the book by Dr. H. Beyer, Theorie und Praxis des Phasenkontrastsverfahrens [Theory and Practice of the Phase Contrast Method], published by Akademische Verlagsgesellschaft of Frankfurt/Main in 1965. Briefly stated, the method consists in imaging a diaphragm aperture, of annular or other preselected geometric shape in the rear focal plane of the condenser of a transmitted-light microscope, on a corresponding annular phase plate in the pupil of the objective employed. The phase-contrast image is produced by interference of the light which passes directly through the object and the phase plate with the light which is diffracted on the object and passes through the objective alongside the phase annulus. A condensed description of this effect can also be found in ABC der Optik, editor Karl Mutze, published by Verlag Werner Dausien of Hanau/Main in 1961, on pages 634 to 638.
In their book Theory and Practice of Scanning Optical Microscopy, Academic Press 1984, T. Wilson and C. Sheppard describe how the Zernicke phase contrast method can also be applied to optical scanning microscopes. This reference utilizes a microscope design which is well-known in conventional microscopy, namely, the design uses a condenser and an annular diaphragm aperture in its rear focal plane. The above-described prior art optical scanning microscope for producing phase-contrast images, shown in the Carl Zeiss publication W41-910d, also uses this same conventional construction.
Copending U.S. patent application Ser. No. 30755 discloses an X-ray microscope which is suitable for producing phase-contrast images. This X-ray microscope has a condenser in the form of a first, so-called zone plate for the irradiating of the object and an objective in the form of a further zone plate with which the object is imaged. A phase-shifting element, which effects the phase contrast in a manner similar to that used in conventional optical microscopy, is arranged on the Fourier plane of the second zone plate.
Scanning microscopes for radiography are also known. In these apparatus, the object is moved in the form of a raster under an X-ray beam which is focused in the form of a spot, and the X-radiation transmitted through the object is directly detected by a detector. As a rule, no condenser is included in X-ray scanning microscopes because the zone plates which would be used as condensers have only very slight diffraction efficiency. Due to the elimination of the condenser, such microscopes can therefore operate with a lower X-ray dosage, i.e., radiation which is gentler on the object.
However, no device for producing phase-contrast images is yet known for scanning X-ray microscopes, since heretofore it has been assumed that a condenser is required for such a method and, accordingly, that it would be necessary to use a higher and undesirably damaging X-ray dose.
It is the object of this invention to create a phase-contrasting method and apparatus which can be used with scanning microscopes to produce comparable phase-contrast images at less expense and with lower radiation intensities.